Synthesis gas (syngas) is a mixture of carbon monoxide (CO) gas, carbon dioxide (CO2) gas, and hydrogen (H2) gas, and other volatile gases such as CH4, N2, NH3, H2S and other trace gases. Syngas is produced by gasification of various organic materials including biomass, organic waste, coal, petroleum, plastics, or other carbon containing materials. Acetogenic Clostridia microorganisms grown in an atmosphere containing syngas are capable of absorbing the syngas components CO, CO2, and H2 and producing aliphatic C2-C6 alcohols and aliphatic C2-C6 organic acids. These syngas components activate Wood-Ljungdahl metabolic pathway 100, shown in FIG. 1, which leads to the formation of acetyl-CoA 102, a key intermediate in the pathway. The enzymes activating Wood-Ljundahl pathway 100 are carbon monoxide dehydrogenase (CODH) 104 and hydrogenase (H2ase) 106. These enzymes capture the electrons from the CO and H2 in the syngas and transfer them to ferredoxin 108, an iron-sulfur (FeS) electron carrier protein. Ferredoxin 108 is the main electron carrier in Wood-Ljungdahl pathway 100 in acetogenic Clostridia, primarily because the redox potential during syngas fermentation is very low (usually between −400 and −500 mV). Upon electron transfer, ferredoxin 108 changes its electronic state from Fe3+ to Fe2+. Ferredoxin-bound electrons are then transferred to cofactors NAD+ 110 and NADP+ 112 through the activity of ferredoxin oxidoreductases 114 (FORs). The nucleotide cofactors (NAD+ and NADP+) are used for the generation of intermediate compounds in Wood-Ljungdahl pathway 100 leading to acetyl-CoA 102 formation.
Acetyl-CoA 102 formation through Wood-Ljungdahl pathway 100 is shown in greater detail in FIG. 2. Either CO2 202 or CO 208 provide substrates for the pathway. The carbon from CO2 202 is reduced to a methyl group through successive reductions first to formate, by formate dehydrogenase (FDH) enzyme 204, and then is further reduced to methyl tetrahydrofolate intermediate 206. The carbon from CO 208 is reduced to carbonyl group 210 by carbon monoxide dehydrogenase (CODH) 104 through a second branch of the pathway. The two carbon moieties are then condensed to acetyl-CoA 102 through the action of acetyl-CoA synthase (ACS) 212, which is part of a carbon monoxide dehydrogenase (CODH/ACS) complex. Acetyl-CoA 102 is the central metabolite in the production of C2-C6 alcohol s and acids in acetogenic Clostridia.
Wood-Ljungdahl pathway 100 is neutral with respect to ATP production when acetate 214 is produced (FIG. 2). When ethanol 216 is produced directly from acetyl-CoA 102, one ATP is consumed in a step involving the reduction of methylene tetrahydrafolate to methyl tetrahydrofolate 206 by a reductase, and the process is therefore net negative by one ATP. The pathway is balanced when acetyl-PO4 218 is converted to acetate 214.
Acetogenic Clostridia organisms generate cellular energy by ion gradient-driven phophorylation. When grown in a CO atmosphere, a transmembrane electrical potential is generated and used to synthesize ATP from ADP. Enzymes mediating the process include hydrogenase, NADH dehydrogenase, carbon monoxide dehydrogenase, and methylene tetrahydrofolate reductase. Membrane carriers that have been shown to be likely involved in the ATP generation steps include quinone, menaquinone, and cytochromes.
The acetogenic Clostridia produce a mixture of C2-C6 alcohols and acids, such as ethanol, n-butanol, hexanol, acetic acid, and butyric acid, that are of commercial interest through Wood-Ljungdahl pathway 100. For example, acetate and ethanol are produced by C. ragsdalei in variable proportions depending in part on fermentation conditions. However, the cost of producing the desired product, an alcohol such as ethanol, for example, could be lowered significantly if the production could be maximized by reducing or eliminating production of the corresponding acid, in this example acetate. Under some fermentation conditions, the production of acetate is best maintained within a range in which sufficient acetyl-PO4 is converted to acetate to produce adequate ATP for the micro-organisms' viability without accumulation of acetic acid. It would therefore be desirable to metabolically engineer acetogenic Clostridia for improved production of selected C2-C6 alcohol s or acids through Wood-Ljungdahl pathway 100 by modulating enzymatic activities of key enzymes in the pathway.